The present invention relates to cell sorter which can be used to analyze and sort cells of interest for bio-medical applications.
Embryonic stem cells possess the ability to remain undifferentiated and propagate indefinitely in culture while maintaining their normal karyotypes and pluripotency to differentiate into the derivatives of all three embryonic germ layers (i.e. endoderm; mesoderm and ectoderm), including such highly specialized cells as neurons, pancreatic and heart cells, etc. that do not normally regenerate in vivo. In vitro differentiation, which is required for therapeutic applications, remains a stochastic process that non-specifically generates all the three germ layers and their derivatives to varying degrees. It is therefore necessary to purify the cell lineage of interest for clinical use. Similarly, adult stem cells for therapeutic applications can be harvested from various tissues of the human body (e.g. bone marrow, adipose tissue, etc.), but the cells need to be isolated and purified for therapeutic applications. Although first used for hematopoietic reconstitution, stem cells are now central to major efforts in regenerative medicine across multiple organ systems, including but not limited to; vascular, myocardial and neuronal repair. Central to all therapeutic efforts that utilize pluripotential stem cells is the ability to rapidly identify and non-destructively isolate individual cells using a cost effective process.
Developing of a cell based screening assay often requires identification and isolation of particular cells from a mixture of various kinds of cells. Moreover, in order to obtain reproducible data on cells and their use in cell-based therapies, reliable and non-destructive purification of cells is essential. Multiple cell isolation and purification techniques are being used in stem cell arena. Currently, magnetic-activated cell separation (MACS) provides a parallel rapid method for cell purification. However, due to the limited specificity of single antibodies in this technique and the omni-present non-specific binding, the purity of cell purification is marginal. It is usually used as a pre-purification method. The cell by cell sorting method currently provides the highest purification rates because multiple signals can be used to increase specificity. Fluorescence activated cell sorters (FACS) using flow cytometry are widely used in research clinics for cell isolation and purification. In a typical flow cytometer (1,2), individual particles pass through an illumination zone, typically at a rate of some 10,000 cells per second, and appropriate detectors, gated electronically, measure the magnitude of a pulse representing the extent of light scattering or fluorescence from labeled antibodies. The FACS instrument combines two basic functions: cell analysis and cell sorting. Fluorescence from labeled antibodies bound to cell surface markers is analyzed on a cell by cell basis in the analysis portion. The cell population of interest is then further sorted into a separate port and accumulated by electrically deflecting the flow stream. The essential character of the flow cytometric approach is strictly quantitative. The large number of available fluorescent antibody tags makes flow cytometry a unique tool for cell analysis and sorting.
FIG. 1 shows the schematic diagram of a conventional flow cytometric cell sorting system 100. A cell 103 passes the interrogation zone 102, where it is excited by laser beam 105 and its light scattering and fluorescence is collected by lens 107 and received by detection system 109. Typically, the flow cell 103 is vibrated at some 10s of kHz to ensure that a uniform stream of droplets emerges from the end of the flow cell. The cell concentration is dilute enough so that the majority of droplets contain either zero or one cell. If a cell or droplet has been identified to be of interest, it is electrostatically charged by 108, causing the droplet to be deflected while passing a system of electrodes 111 and 112. The sorted cells 113 and 114 are collected by the collection tubes 115 and 116, respectively. Since the fluid with cells 103 is moving at a rate of 1 to 10 ms/droplet and the distance from the flow cell interrogation zone 102 to the deflector formed by the electrodes 111 and 112 is about 5 mm, the sorting decision needs to be made in less than 0.5 to 5 ms, allowing the sorting of some thousands of cells per second.
However, the rather bulky and complicated nature of the instrumentation as shown in FIG. 1 leads to fairly low adaptation rates in clinical labs. It is very difficult for a clinical lab to obtain appropriate approvals necessary to certify the cleanliness due to patient sample contamination in flow system for therapeutic applications. Current cytometer require careful and extensive cleaning procedures or exchanging of their sample handling components for this application, which requires a highly trained technician and can take many hours between running samples.